The present invention relates to a novel polymeric resin and method suitable for use in creating polypeptides.
Solid phase peptide synthesis (SPPS) has proven to be the only practical method of producing synthetic peptides longer than 8-10 amino acids. One method of producing synthetic peptides is called Boc (tert-butyloxycarbonyl) SPPS. Another method of producing synthetic peptides is known as Fmoc (9-fluorenylmethyloxy carbonyl) SPPS. Both Boc and Fmoc SPPS employ minuscule spheres of specially produced resin which are suspended in a chemical solution. Each of these spheres serve as a anchor for the assembly of amino acid chains to produce polypeptides.
The Boc SPPS, although the most popular currently used method for the synthesis of peptides, suffers from low yield of product (especially with peptides of medium to long length), low purity, and an inability to build certain combinations of the amino acids except with great difficulty and expense. Specifically, trifluoroacetic (TFA), normally used to deprotect the amino terminal during each synthesis cycle in the Boc protection strategy, also cleaves approximately 1% of the resin anchor peptides from the resin spheres. Thus, yield and purity diminishes rapidly during the synthesis processes. The latter is a result of cleaved and lost chains leaving free initiation sites on the resin, thus permitting new chains to start building thereupon. Moreover, hydrofluoric acid (HF), an integral component of Boc SPPS, is responsible for many destructive side effects when applied to synthetic peptides.
Fmoc SPPS improves upon Boc SPPS in that an orthogonal synthesis system is employed. In other words, the side-chain blocking groups and the peptide-resin linkages are completely stable to the agents used during each step of the synthesis of peptides. Specifically, the deprotecting step of each cycle employs a secondary amine base which does not attack the bond between the growing peptide chain and resin support, since this bond is acid labile. Moreover, only TFA is used with Fmoc, eliminating the destructive side effects of HF. Some Fmoc SPPS, however, employs polyamide resin which generally possesses a low loading capacity. Present polyamide resins also tends to clump together rendering it impotent for large scale production.
Reference is made to U.S. Pat. Nos. 4,831,084 and 4,914,151 to Mergler et al which describes polystyrene resin-linker formulations which are usable in Fmoc SPPS. Unfortunately, the resin described in Mergler is difficult to prepare since the starting material must made by a multi-step reaction. In addition, the finished synthetic peptide is cleaved from the resin employing 1% TFA in dichloromethane (DCM). It has been found that 1% TFA is strong enough to remove a significant fraction of the side chain blocking groups, either Boc or triphenylmethyl (Trt). Boc is used to block the functional moiety on the side chains of the Lys, while Trt blocks Cys and His. Elimination of these amino acids from any peptide construction severely hinders applicability of the same. Also, the Mergler resin generally operates at a low level of substitution, thus, precluding large scale industrial processing. Making such resin with high loading characteristics, would be enormously costly since a starting material for this resin is expensive.
Other resins developed for the production of protected peptide segments or fragments by Fmoc-SPPS were susceptible to similar problems. Most notably, prior resins flaws include the irreversible attachment of tryptophan to the support resin and the alkylation of various amino acids when the product is being cleaved from the solid support matrix.
There has been a continuing need for resins usable in Fmoc SPPS which result in higher yields, and higher purity, and which are applicable to the building of all sequences of both naturally occurring and unnatural amino acids, as well as other molecules containing carboxylic acids moieties.